Microsoft Word - Akkaldeo Mishra.doc A. D. Mishra / BIBECHANA 8 (2012) 17-22 : BMHSS, p. 17 BIBECHANA A Multidisciplinary Journal of Science, Technology and Mathematics ISSN 2091-0762 (online) Journal homepage: http://nepjol.info/index.php/BIBECHANA A Convenient route for the synthesis of antimicrobial 1, 3, 4- thiadiazole derivatives A. D. Mishra Dept. of Chemistry, Tribhuvan University, P. N. Campus, Pokhara Ph. No.: 061-540414 / Mob, No.: 9846027853 e-mail: mishraad05@hotmail.com Article history: Received 18 May, 2011; Accepted 17 June, 2011 Abstract Thiosemicarbazide of 6-chloro-2- aminobenzothiazole on cyclization with different aromatic carboxylic acids in Phosphorus oxychloride provided the corresponding 2-aryl-5-(6’-chloro-1’,3’- benzothiazole-2-yl-amino)-1,3,4-thiadiazoles 4a-j. The compounds are characterized by elemental analysis, IR and 1 H NMR spectral data. All the compounds are evaluated in vitro for their antimicrobial activities against several fungal and bacterial strains and showed significant activities. Keywords: Aminobenzothiazole; 1, 3, 4- thiadiazole; antimicrobial; cyclization 1. Introduction Benzothiozoles are bicyclic ring systems with diverse chemical reactivity and broad spectrum of biological activities such as antimicrobial[1,2],antitumor[3,4],anti-inflammatory[5,6]and antilieshmanial[7,8], etc. Several 1, 3, 4- thiadiazole derivatives are also known to exhibit diverse biological properties like antimicrobial [9,10] , antitubercular[11,12] , anti-inflammatory[13] and anticonvulsant[14]. The synthesis and biological activities of a few benzothiozole derivatives linked with 1,3,4-thiadiazole systems is reported here owing to their pharmaceutical importance. The reaction sequence leading to the formation of the desired heterocyclic compounds are outlined in scheme 1.The 6-chloro-1,3-benzothiazol-2-yl-thiosemicarbazide 2 was prepared by the reaction of 6-chloro-2-aminobenzothiazole 1 with CS2 and hydrazine in the presence of ethanol. Several 2-aryl-5-(6’-chloro-1,’3’-benzothiazol-2-yl-amino)- 1, 3, 4- thiadiazoles 4a-j were synthesized by the reaction of 6-chloro-1,3-benzothiazol-2-yl-thiosemicarbazide 2 with various substituted aromatic carboxylic acids 3a-j in the presence of phosphorous oxychloride. 2. Material and Methods Melting points were measured in open capillary tubes and are uncorrected. IR spectra were recorded on a Nicolet, 5PC FT-IR spectrometer in KBr pelletes and 1 H NMR spectra on the bruker DRX-300 FT NMR spectrometer using TMS as internal reference.Chemical shifts are observed with DMSO-d 6 solvent in ppm. The purity of the compounds was checked on silica gel A. D. Mishra / BIBECHANA 8 (2012) 17-22 : BMHSS, p. 18 G plates using iodine vapor as visualizing agents. Ofloxacin was used as standard dug for antibacterial activity ketoconazole as standard drug for antifungal activity [15,16]. The precursor 6-chloro-2- aminobenzothiazole 1 was synthesized by the already reported procedure [17]. Scheme 1 N SCl NH2 CS2 C2H5OH NH2 Cl N S NH C S NH RCOOHPOCl3 Cl N S NN S NH R 1 2 NH2NH2 a-j3 4a-j ( ) ( ) ( ) ( ) R = a. Phenyl b. 4-Chlorophenyl c. 2,4-Dichlorophenyl d. 4-Nitrophenyl e. 2-Aminophenyl f. 2,4-Dichlorophenoxymethyl g. 2-Naphthylmethyl h. 4-methoxyphenyl i. 2-Acetoxyphenyl j. 3-Pyridyl 2.1 General Procedure for the Synthesis of 6-Chloro-1,3- benzothiazol-2-yl- thiosemicarbazide (2) The 6-chloro-2- aminobenzothiazole 1 (0.01 mole) was dissolved in ethanol (90%, 50ml) and then CS2 (20ml) was added slowly within 10 minutes with constant shaking. The resulted solution was then allowed to stand for 1.0 hr. Then 20 ml of 50% hydrazine solution was added to it. The reaction mixture was warmed slowly for 1.30 hr then filtered, washed with cold water and dried. The solid thus obtained was purified by recrystallization from methanol. 2.2 General Procedure for the Synthesis of 2-Aryl-5-(6’-chloro-1’, 3’- benzothiazol-2-yl- amino)- 1,3,4-thiadiazoles (4a-j) A mixture of 6-chloro-1,3- benzothiazol-2-yl- thiosemicarbazide 2 (0.01 mole), an aromatic acid 3a-j (0.01 mole) and phosphorous oxychloride (25 ml) was refluxed for 3 hr. After cooling to room temperature the reaction mixture was slowly poured over crushed ice and kept for 1 hr. The solid thus separated was filtered, washed with cold water, dried and purified by recrystallization from methanol. 3. Results and Discussion All the synthesized thiadiazole derivatives 4a-j are confirmed by different spectral analysis (table 2).These compounds have given similar IR and 1 H NMR signals for main moiety with variations for substitutions. The characteristic observations for the substitutions have further supported the formation of mentioned products 4a-j.The IR spectrum of the typical compound 4i showed absorption peak at 1710 cm -1 due to the stretching of C=0. The N-H stretching vibration appeared at 3436 cm -1 . The absorption at 795 cm -1 was obtained due to C-Cl stretching vibration. The 1 H A. D. Mishra / BIBECHANA 8 (2012) 17-22 : BMHSS, p. 19 NMR spectra of this compound displayed on singlet at δ 2.13 showing the presence of OCCH3 protons of acetyl group. A broad singlet appeared at δ 12.36 showing the presence of NH proton. The seven aromatic protons of benzothiozole and phenyl ring were observed as a multiplet at δ 7.5-8.0. Table 1: Physical characterization data of compounds (4a-j) Comp.no R Yield (%) m.p.(˚C) 4a Phenyl 65 225 4b 4-Chlorophenyl 61 270 4c 2,4-dichlorophenyl 67 283 4d 4-Nitrophenyl 72 285 4e 2-Aminophenyl 66 227 4f 2,4-dichlorophenoxymethyl 58 215 4g 2-Napthylmethyl 73 165 4h 2-Methoxyphenyl 61 201 4i 2-Acetoxyphenyl 60 272 4j 3-Pyridyl 55 242 4. Biological Evaluation All the compounds have been screened for both antibacterial and antifungal activities using cup- plate agar diffusion method by measuring the inhibition zone in mm. Ofloxacin(50 µg/ml) was used as a standard drug for antibacterial activity and ketoconazole(50 µg/ml) as a standard drug for antifungal activity. The compounds 4a-j were screened for antibacterial activity against E. coli, S. aureus and P. aerugenosa in nutrient agar medium and for antifungal activity against A. niger and C. albicans in sabouraud’s dextrose agar medium. These sterilized agar media were poured into petri-dishes and allowed to solidify. On the surface of media microbial suspensions were spreaded with the help of sterilized triangular loop. A presterilized stainless steel cylinder of 8 mm diameter was used to bore cavities. All synthesized compounds (50 µg/ml) were placed serially in the cavities with the help of micropipette and allowed to diffuse for 1.00 hr. DMF was used as a solvent for all the compounds and as a control. These plates were incubated at 37˚C for 36 hr. and 28˚C for 48 hr, for antibacterial and antifungal activities respectively. The zone of inhibition observed around the cups after respective incubation was measured and percentage inhibition of A. D. Mishra / BIBECHANA 8 (2012) 17-22 : BMHSS, p. 20 the compounds was calculated (tables 3&4).The thiadiazole derivative 4i having acetoxyphenyl group showed potent activity against S. aureus(92%), wereas compound 4g having 2- nepthylmethyl group showed maximum inhibition against E. coli(95%), when compared to standard drug ofloxacin. The compound 4c having 2,4-dichlorophenyl group also showed significant antibacterial activity(85, 87 and 82%) against S. aureus, E. coli, and P. aerugenosa respectively. Rest of the compounds showed moderate to good antibacterial activity. Table 2: Spectral data of the compounds (4a-j) Comp.no. IR, ν (cm -1 ) 1 H NMR, δ (ppm) 4a 3360(N-H) 3030(C-H) 750(C-Cl) 7.35-7.8(m,8H,ArH) 8.27(S,1H,NH) 4b 3369(N-H) 3032(C-H) 755(C-Cl) 7.30-7.96(m,7H,ArH) 10.48(S,1H,NH) 4c 3386(N-H) 3060(C-H) 765(C-Cl) 7.34-7.62(m,6H,ArH) 10.50(S,1H,NH) 4d 3366(N-H) 3033(C-H) 752(C-Cl) 7.37-8.38(m,7H,ArH) 13.25(S,1H,NH) 4e 3411(N-H) 3033(C-H) 752(C-Cl) 4.5(S,2H,NH2) 7.08-7.86(m,7H,ArH) 12.56(S,1H,NH) 4f 3408(N-H) 3028(C-H) 750(C-Cl) 4.97(S,2H,CH2O) 7.06-7.89(m,6H,ArH) 12.59(S,1H,NH) 4g 3402(N-H) 3021(C-H) 752(C-Cl) 3.27(S,2H,CH2) 7.50-7.87(m,10H,ArH) 12.34(S,1H,NH) 4h 3399(N-H) 3032(C-H) 752(C-Cl) 3.86(S,3H,OCH3) 7.05-7.86(m,7H,ArH) 12.56(S,1H,NH) 4i 3436(N-H) 3038(C-H) 1710(C=O) 795(C-Cl) 2.13(S,3H,OCH3) 7.5-8.0(m,7H,ArH) 12.36(S,1H,NH) 4j 3421(N-H) 3036(C-H) 745(C-Cl) 7.05-7.89(m,7H,ArH) 13.0(S,1H,NH) A. D. Mishra / BIBECHANA 8 (2012) 17-22 : BMHSS, p. 21 Table 3: Antibacterial activity of 1, 3, 4-thiadiazole derivatives (4a-j) Comp. Staphylococcus aureus Escherichia coli Pseudomonas aerugenosa Zone of inhibition (mm) % inhibition Zone of inhibition (mm) % inhibition Zone of inhibition (mm) % inhibition 4a 10 65 9 63 13 77 4b 12 80 10 69 12 70 4c 15 85 16 87 14 82 4d 11 76 14 85 12 71 4e 11 80 13 71 11 74 4f 14 82 12 65 13 76 4g 13 76 16 95 13 81 4h 10 78 12 75 12 70 4i 16 92 13 72 14 83 4j 12 80 11 69 13 79 Table 4: Antifungal activity of 1, 3, 4- thiadiazole derivatives (4a-j) Aspergillus niger Candida albicans Comp. Zone of inhibition (mm) % inhibition Zone of inhibition (mm) % inhibition 4a 20 62 12 66 4b 14 55 11 58 4c 13 47 13 62 4d 11 45 11 61 4e 25 87 15 78 4f 23 82 16 83 4g 16 56 14 67 4h 12 46 13 69 4i 13 48 12 63 4j 15 52 14 71 A. D. Mishra / BIBECHANA 8 (2012) 17-22 : BMHSS, p. 22 The results of antifungal activity of the test compounds were quite different from their antibacterial activity. The 1,3,4- thiadiazole derivative 4e having 2-aminophenyl group showed maximum inhibition(87%) against A. niger whereas 4f having 2,4-dichlorophenoxymethyl groups showed maximum inhibition (83%) against C. albicans. Furthermore 1, 3, 4- thiadiazole dericatives 4e showed 78% inhibition against C. albicans. Whereas 4f showed 82% inhibition against A. niger. Rest of the thiadiazole derivatives showed moderate to good antifungal activity as compared to the reference drug ketoconazole. Thus it is concluded from the screening results that 1,3,4- thiadiazole derivatives 4a-j have been proved to be more effective against all the tested microorganisms at a concentration of 50 µg/ml. 5. Conclusion The reaction of 6-Chloro-2- aminobenzothiazole 1 with hydrazine and carbon disulphide in alcoholic medium yielded corresponding thiosemicarbazide 2 in excellent yield of 82% within 1 hr. Thus obtained thiosemicarbazide produced various 1,3,4-thiadiazoles 4a-j, through cyclization upon reaction with carboxylic acids 3a-j in presence of phosphorous oxychloride in 75% yield within 3 hr. All the synthesized thiadiazoles 4a-j, showed excellent to moderate antifungal and antibacterial activities, which has proved the thiadiazoles 4a-j as pharmaceutically valuable heterocycles. This synthetic method is proved to be simple, easier to workup and environment friendly tool in pharmaceutical chemistry. Acknowledgements The auther is thankful to the Research Committee, Department of Chemistry and Department of Microbiology, Tribhuvan University,P. N. Campus, for providing miniresearch grant and available laboratory facilities regarding synthesis and microbial activities of the compounds. Thanks are due to the Instrumental Laboratory, Department of Chemistry, University of Delhi, for assisting in spectral analysis of all the synthesized compounds. References [1] I. Yildiz-Oren, I. 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